1. Field of the Invention
The invention relates to methods for screening compounds for their ability to modulate actin polymerization. The invention is also related to compounds which modulate actin polymerization.
More specifically, the invention is related to methods of screening compounds which modulate the interaction of cortactin or HS-1 with Arp2/3. The invention is also related to compounds which modulate the interaction of cortactin or HS1 with Arp2/3. Also provided are mutants of cortactin with decreased ability to interact with Arp2/3.
2. Related Art
Amplification of the chromosome 11q13 constitutes an important genetic mechanism to progression of a subset of cancers, including breast cancer and head and neck carcinomas (Schuuring, E. D., et al., Mol. Cell Biol. 13:2891–2898 (1993)). The amplification often results in overexpression of cortactin, a filamentous actin (F-actin) associated protein. (Wu, H., et al., Mol. Cell Biol. 11:5113–5124 (1991); Zhan, X., et al., J. Biol. Chem. 268:24427–24431 (1993)). While overexpression of cortactin is usually correlated with poor prognosis (Schuuring, E., Gene 159:83–96 (1995)), presumably due to enhanced metastasis, the biochemical and biological functions of cortactin remain unclear.
Cortactin is a prominent substrate for several non-receptor protein tyrosine kinases, including Src, Fer and Syk (Wu, H., et al., Mol. Cell Biol. 11:5113–5124 (1991); Maruyama, S., et al., J Biol. Chem 271:6631–6635 (1996); Kim, L., and Wong, T. W., J. Biol. Chem. 273:23542–23548 (1998); Gallet, C., et al., J. Biol. Chem. 274:23610–23616 (1999)). Tyrosine phosphorylation of cortactin is a common cellular response to growth factors, stress, cell shrinkage, and cell injury mediated by reactive oxygen radicals (Zhan, X., et al., J. Biol. Chem. 268:24427–24431 (1993); Liu, M., et al., J. Biol. Chem. 271:7066–7071 (1996); Kapus, A., et al., J. Biol. Chem. 274:8093–8102 (1999); Li, Y., et al., (in press)). In vitro, tyrosine phosphorylation mediated by Src decreases the F-actin cross-linking activity of cortactin. (Huang, C., et al., J. Biol. Chem. 272:3911–13915 (1997)).
Cortactin has a unique structure featured by six and one half tandem repeats of 37-amino-acid sequence and a carboxyl terminal SH3 domain (FIG. 1A). Between the repeat and the SH3 domain there are a proline-rich sequence, an alpha helical region and three tyrosine residues that are targeted by Src-related kinases (Huang, C., et al., J. Biol. Chem. 273:25770–25776 (1998)). In addition, cortactin contains an N-terminal domain, which is conserved across species and in HS1, a cortactin-related protein that is exclusively expressed in the hematopoietic lineage (Kitamura, D., et al., Nucleic. Acids. Res. 17:9367–9379 (1989)).
In cultured fibroblasts, cortactin is mainly distributed within cell leading edges such as lamellipodia and punctate-like structures (Wu, H., et al., Mol. Cell Biol. 11:5113–5124 (1991); Huang, C., et al., J. Biol. Chem. 273:25770–25776 (1998)). In MDA-MB-231 cells, an invasive breast cancer cell line, cortactin is associated with invadopodia, cortical structures that penetrate into and degrade extracellular matrix during invasion (Bowden, E. T., et al., Oncogene 18:4440–4449 (1999)).